This application is submitted in response to PA-11-159: Biomarkers of Infection-Associated Cancers. The objective of this study is to identify a biomarker specific for Human Papillomavirus-associated Head and Neck Squamous Cell Carcinoma (HPV-HNSCC) in order to develop a sensitive and specific screening tool to detect early stage cancers as well as to identify subpopulations of HPV-exposed individuals who may be at increased risk for developing HPV-HNSCC. We plan to achieve this goal by evaluating methylation of HPV genes in Oral Rinse Samples (ORS) of HPV-HNSCC patients. Integration of viral DNA into the host genome is one of the first molecular steps that occurs in the progression to cancer due to the inadvertent deletion of viral regulatory genes. After viral integration, the HPV genome also becomes susceptible to modification by the host epigenetic machinery. Therefore, the detection of methylated HPV genes indicates viral integration has occurred. Since viral integration can lead to cellular transformation, the detection of integrated, or methylated, viral genes in ORS can be a useful biomarker to diagnose early stage cancers and/or to identify HPV-exposed individuals at increased risk for developing head and neck cancer. Methylation-specific PCR (MSP) is an established method to detect methylated genes in tissue and body fluids. The detection of methylated HPV genes in the ORS via MSP may be a novel screening tool that we propose to develop in this study. When developing biomarkers, the success rate is often improved through the identification and screening of those subjects at highest risk for developing cancer. The partners of patients diagnosed with HPV-HNSCC are individuals at risk for persistent oral HPV- 16 exposure and infection, which is a known risk factor for the development of HPV-HNSCC. Therefore, we plan to evaluate for HPV gene methylation in the ORS of both HPV-HNSCC patients and matched partners. We will compare the frequency of detection of methylated, or integrated, HPV-16 genes in the ORS of patients, matched partners, and non-cancer controls to determine if methylated viral genes, such as L1, can serve as a biomarker for HPV-HNSCC and to determine if the partners of HPV-HNSCC patients represent a subpopulation of HPV-exposed individuals who may be at increased risk for developing HPV-HNSCC due to a higher frequency of persistent, or integrated, oral HPV infection as compared to a matched control group. The ORS from patients, partners, and matched controls already have been collected and are ready to be used for this proposed study.